HPV Vaccine Comprising Peptides From Host Cell Proteins

ABSTRACT

The present invention relates to a human papillomavirus (HPV) vaccine that comprises peptides from host cell proteins and more particularly, a vaccine that is directed against cancers that are associated with HPV infections, such as cervical cancer, head and neck cancer and skin cancers. The peptides comprise fragments of host cell proteins that have been targeted for degradation by HPV proteins, such as E6 and E7 and are presented on the surface of HPV infected cells in relatively large amounts. These peptides can be recognised by CTL and elicit an immune response, and are therefore ideal tumour-specific markers. The invention also relates to novel peptide: peptide complexes such as peptide/HLA complexes and their use in a tumour-specific vaccine.

This application is a continuation utility patent application from U.S. Utility patent application Ser. No. 11/671,095, which in turn is a continuation of international application number PCT/GB2005/002962 (publication no. WO 2006/013336) filed on Jul. 27, 2005 entitled HPV VACCINE COMPRISING PEPTIDES FROM HOST CELL PROTEINS, and claims the benefit of priority in the above-mentioned United States and international applications, and also the benefit of priority in the corresponding British national patent application no. GB 0417430.6 filed on Aug. 5, 2004 and entitled A NOVEL HPV VACCINE COMPRISING PEPTIDES FROM HOST CELL PROTEINS, the disclosures of each of which are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to a human papillomavirus (HPV) vaccine that comprises peptides from host cell proteins and more particularly, but not exclusively, to a vaccine that is directed against cancers that are associated with HPV infections, such as cervical cancer, head and neck cancer and skin cancers. The peptides comprise fragments of host cell proteins that have been targeted for degradation by HPV proteins, such as E6 and E7. Further, the invention relates to the identification of novel peptides and uses thereof. Additionally, the invention relates to novel peptide: peptide complexes and uses thereof.

BACKGROUND TO THE INVENTION

Human papillomavirus (HPV) is a very common virus that causes abnormal growth of tissue on the feet, hands, vocal cords, mouth and genital organs. Over 60 types of HPV have been identified and each type infects certain parts of the body. HPV is mainly spread through physical contact with an infected individual. In the majority of cases, HPV disappears within 1-2 years and indeed, during the course of the infection, may be subclinical; the individual may be unaware of their infection. However, in a small number of cases, HPV can progress and develop into cancer.

There are two kinds of abnormal tissue caused by HPV: condyloma (warts) and dysplasia (pre-cancer). Wart-like growths can be found in any infected areas and may cause itching, burning or slight bleeding. In these instances, antiviral creams may be prescribed or, in some cases, the growth may be removed or destroyed by cold cautery (freezing that destroys tissue) or hot cautery (burning warts off with an electric instrument or laser treatment).

Where HPV infection progresses to cancer, cancer patients are treated by a combination of surgery, radiotherapy and chemotherapy. However, radiotherapy and chemotherapy have the disadvantage of destroying healthy as well as malignant cells, and can thus cause severe side effects, while surgery is invasive and leaves the patient open to secondary infections. These side effects and risks are undesirable, and coupled to this is the fact that these treatments are not always successful, resulting in the majority of patients entering relapse and so representing with the disease.

It is therefore clear that more effective treatments are required, and it has been suggested that the specificity of the immune system might be harnessed against virally infected cells. This concept has been termed “immunotherapy”.

In particular, it has been shown that cancer patients have T cells that are capable of recognising their tumour cells, but these cells do not divide and differentiate into cytotoxic T lymphocytes (CTL) which are capable of killing these cells.

Cytotoxic T lymphocytes kill “target” cells, such as virally-infected cells, and have also been implicated in the “immune surveillance” of cancer cells. The majority of CTL belong to the CD8⁺-subset of T cells and have T-cell receptors (TCR). These TCR are able to recognise peptides when they are expressed on the surface of cells in association with class 1 major histocompatibility complex (MHC) molecules. In man, each class of MHC is represented by more than one locus; these are called human leucocyte antigen (HLA). The class 1 HLA loci are HLA-A, -B, -C, -E, -F and -G. Additionally each HLA has different alleles and Table 1 lists those alleles that have been identified to date.

When a CTL encounters an antigen/MHC complex for which its TCR is specific, it enters the cell cycle and goes through several rounds of mitosis, followed by differentiation into an effector/killer cell. Differentiation includes forming a large number of modified lysosomes that contain the cell-killing proteins perforin and granzyme. Once the CTL have killed the target cells most of them will die, although a small proportion become memory cells that can respond to the antigen quickly if it reappears.

Tumour-reactive cytotoxic T lymphocytes have been shown to mediate tumour regression in animal models (1) and in man (2), and there has thus been an interest in using tumour-specific CTL's as an immunotherapy for human cancers.

In this regard monoclonal antibodies have been shown to be effective against some cancers, especially cancers of white blood cells, and are targeted at a molecule or receptor that is associated with cancer cells. Table 2 lists some of these antibodies and their mechanism of action.

Alternatively, dendritic-cell vaccines have been used to elicit a tumour-specific CTL response. Dendritic cells are the most potent antigen-presenting cells and they act by engulfing antigen, processing it into peptides and presenting it to T cells. To make a dendritic-cell vaccine, dendritic cells are harvested, exposed in vitro to antigen associated with the type of tumour in the patient, and then re-injected into the patient. To date these vaccines have shown some promise against melanoma, prostrate cancer and lymphoma.

Ideally these vaccines target molecules that are expressed on cancer cells, but not on healthy cells. However such tumour-specific antigens have been hard to find, and as a result many of the immune agents now in use also target healthy cells in the hope that these cells, eventually, will be replaced. As with radiotherapy and chemotherapy, this treatment can cause severe side effects and also leads to the potential for autoimmunity (3). Indeed, in the case of a telomerase vaccine, this protein is also present in the stem cells of bone marrow, reproductive organs and perhaps other tissues. Further, the antigen to which some dendritic cells are exposed include tyrosinase, which is to be found in melanocytes, or prostatic acid phosphatase (PAP), which is to be found in prostate cells.

It is therefore clear that additional viral therapies are needed, particularly for those patients with an advanced stage disease that has failed to respond to conventional viral or cancer treatments.

Recently, a number of studies have shown that high-level expression of certain proteins in tumour cells is sufficient to allow CTL to discriminate between tumours and normal cells (4,5).

One way of avoiding autoimmunity in tumour immunotherapy is to target the 15% of human malignancies that are associated with viruses. Of these the strongest association is between cervical cancer and human papillomarivus, with 99.7% of cervical cancers containing HPV DNA (6). There are over 25 HPVs that infect the genital mucosa and give rise to malignancies such as cervical cancer, head and neck cancers and skin cancers. These “high risk” HPVs are characterised by at least two oncogene products: E6 and E7, which act to immortalise and transform, in the cervix, epithelial cells. The expression of these proteins is thought to be essential to retain the transformed phenotype of the cancer cell and so these non-self viral proteins are therefore attractive targets for CTL mediated immunotherapy.

CTL active against HPV E6/E7 can be induced by vaccination (7) and such CTL have been detected with variable frequency in patients with premalignant cervical disease (8) or cancer (9). However it has been difficult to generate these CTL in vitro, probably because they occur at low frequency (10). A major limitation of using these proteins as tumour-specific targets is that they are expressed at low levels in cancer cells (11). Furthermore, the E6 and E7 proteins themselves are small and contain few epitopes suitable for recognition by CTL (12).

The present invention aims to overcome these problems by identifying and then targeting peptides that are recognised by CTL, which peptides are specific to HPV transformed cells and are very unlikely to give rise to autoimmunity. These peptides are either uniquely presented or over-presented in HPV transformed cells, and the proteins from which these peptides are derived are, typically, either absent or appear to be expressed at very low levels in HPV transformed cells. In contrast, these proteins occur at normal or high levels in normal cells.

The invention is based on the mechanism that HPV E6 and E7 oncoproteins use to mediate targeted degradation of host cell proteins such as retinoblastoma proteins (Rb), C-MYC and HMCM7, among others (see Table 3), which takes place during transformation of the infected cell.

It is well known that HPV oncoproteins bind to and facilitate the degradation of host cell proteins, such as Rb. Thus, analysis of HPV transformed cervical carcinomas reveals no apparent expression of full-length Rb protein, whereas normal cells have high cellular levels of the Rb protein, as this is not normally proteolytically degraded (13).

It has been shown that Rb proteins are degraded by the ubiquitin-dependant proteolysis system (13), and more recently, it has come to light that intracellular organelles called proteasomes play a role in mediating degradation (18,19) of host cell proteins after interaction with E6 or E7 oncoproteins.

We have recognised the fact that the degradation of, for example ubiquinated protein substrates by proteasomes, is possibly the major mechanism by which peptides recognised by CTL's are generated (20, 21). For example, in a virally infected cell, newly synthesised viral proteins in the cytoplasm are degraded by proteasomes into peptide fragments. These peptides are transported into the endoplasmic reticulum (ER) by transporter associated with antigen processing (TAP) proteins. Once inside the ER, the peptides will bind to free MHC class I molecules and beta 2 microglobulin to form a mature MHC/peptide complex. This is transported to the cell surface where it may be recognised by CTL. FIG. 1 shows a diagrammatic representation of this process.

Accordingly, the present invention is based on the theory that in HPV transformed cells, Rb proteins (and other proteins, see Table 3) will be targeted for degradation, processed and peptides thereof will be presented on the surface of the cell as peptides that can be recognised by CTL. In non-HPV transformed, or normal, cells these proteins will not be degraded significantly, so these peptides, effectively, will not be available for CTL recognition. Thus, HPV transformed cells should have high levels of, for example, Rb derived peptides typically co-presented on the cell surface in a peptide HLA complex, but low intracellular levels of the full-length proteins, contrary to normal cells (FIG. 2).

The use of host cell proteins as targets for immunotherapy is not novel. However, in all previous instances this approach has relied on the over-expression of proteins in tumours, compared to normal cells. For example, host cell proteins such as p53 (5), Wilms transcription factor (WT1), Her 21Neu (16) and hTert (17) have been proposed as “tumour-specific” antigens, as all of these are over-expressed in tumour cells. To our knowledge, this is the first time that a HPV or cancer vaccine has been directed at “tumour-specific” proteins, and more particularly peptides thereof, that are expressed at normal, low, or undetectable levels in HPV transformed cells, compared to normal cells.

Previously, high levels of antigen expression were thought advantageous in order to allow CTL to discriminate between tumour cells and normal cells.

Additionally, up until now HPV vaccines have comprised proteins that are produced by HPV, not host proteins that are targeted for degradation by this virus.

In summary, the current invention relies on a relatively high level of presentation of peptides at the cell surface but not necessarily on relatively high levels of expression, or apparent expression, of the corresponding proteins in the virally-infected cell. In fact, low level or no expression of the tumour-specific protein would typically indicate that the protein was being targeted for degradation by viral proteins and so was present at low intracellular levels, but following degradation, presented at the cell membrane and so was available as a peptide for CTL recognition.

Accordingly, in one aspect of the invention, there is therefore provided a vaccine comprising: at least one isolated, purified, synthesised or recombinant peptide, wherein the peptide is a fragment of a host cell protein that has been degraded by human papillomavirus oncoproteins, and can elicit a CTL response when administered to a mammal.

Reference herein to a cell protein that has been degraded by human papillomavirus oncoproteins includes reference to a protein that has been selectively targeted for degradation by HPV oncoproteins and so includes a protein that, in a HPV transformed cell, would be selectively targeted for degradation or a protein that is acted upon by human HPV oncoproteins in such a way that it is, directly or indirectly, degraded, most typically but not exclusively, by the ubiquitin pathway.

In a preferred embodiment of the invention the mammal is human.

Preferably, the oncoprotein is E6 or E7.

The host cell protein may be any protein that is degraded by viral proteins, such as E6 or E7, and Table 3 lists those proteins that are currently known to be targeted for degradation by E6 or E7.

Preferably, the peptide is HPV-specific or tumour-specific, meaning that it is presented in high amounts on the cell surface of HPV transformed or tumour cells, relative to normal cells.

Even more preferably, the peptide is 9 to 30 amino acids in length.

Alternatively, the peptide may be 9 to 11 amino acids in length.

The CTL response is preferably a HPV-specific or tumour-specific CTL response, meaning that the CTL can recognise HPV transformed cells or tumour cells expressing the peptides of the vaccine.

More preferably, the vaccine comprises one or more of the peptides shown in Table 4 (SEQ ID NOS: 1-163).

More preferably still, the vaccine comprises any of the aforementioned peptides plus a further protein or peptide comprising a major histocompatability complex molecule, ideally, a class I molecule and more specifically a human leucocyte antigen (HLA), and more ideally still a HLA selected from Table 1.

In another aspect of the invention, there is provided a vaccine comprising: at least one isolated, purified, synthesised or recombinant peptide, wherein the peptide is chosen from those listed in Table 4.

In yet another aspect of the invention, there is provided a vaccine comprising: at least one isolated, purified, synthesised or recombinant peptide selected from Table 4 and, further at least one isolated, purified, synthesised or recombinant HLA selected from those listed in Table 1.

In a further aspect of the invention, there is provided a vaccine comprising: at least one isolated, purified, synthesised or recombinant nucleic acid molecule encoding any peptide or peptide/HLA complex as described above.

In this embodiment, the nucleic acid molecule may be in the form of a vector that comprises a recombinant construct. Ideally the construct is adapted for the expression of said vaccine in a selected host system. The host system is a cell, plasmid, virus, live organism or other similar vehicle.

According to a further aspect of the invention there is provided a host cell transformed or transfected with the vector of the invention.

Additionally, the present invention provides a method of manufacturing a vaccine, which method comprises; culturing a host cell transformed or transfected with a vector comprising a recombinant construct as described above; and isolating/purifying the resulting construct product.

The peptides of the present invention may also be used to generate and isolate HPV-specific or tumour-specific cytotoxic T lymphocytes or their T cell receptors or the genes encoding said receptors, in vitro, for use in adoptive immunotherapy. This could for example be carried out by culturing T lymphocytes with at least one of the peptides described above.

According to yet a further aspect of the invention there is provided a method of identifying HPV-specific or tumour-specific cytotoxic T lymphocytes comprising:

(a) culturing a sample containing cytotoxic T lymphocytes with at least one peptide that represents a fragment of a host cell protein which is degraded by HPV proteins when said host cell is transformed or transfected by HPV whereby said peptide is ultimately presented on a surface of a virally infected cell; and (b) selecting CTL that recognise said peptide by binding thereto.

In a preferred method of the invention said peptide is one selected from the list shown in Table 4. In yet a further preferred method of the invention the CTL are CD8⁺ cells.

It will be apparent to those skilled in the art that CTL receptors may further be identified using the aforementioned method.

The present invention can be used to treat HPV associated diseases and particularly cancer, preferably cervical cancer, head and neck squamous cell cancer, non-melanoma skin cancers, liver cancer, mesothioloma or prostrate cancer.

Furthermore, the present invention also provides a method of treatment, which method comprises administering a vaccine as described above, to a mammal to be treated. Ideally, the mammal is human.

According to a further aspect of the invention there is provided a peptide, or a nucleic acid molecule encoding same, selected from the list shown in Table 4.

In a further embodiment of the invention, said peptide is for use as a vaccine and in particular for use as a HPV vaccine to treat HPV associated disorders.

According to a further aspect of the invention there is provided a complex comprising at least one of the peptides listed in Table 4 in association with a HLA co-presenting peptide.

More preferably the HLA peptide is one of the peptides listed in Table 1 and more specifically HLA-A binding protein and more specifically still HLA-A 0201.

According to a further aspect of the invention there is provided the use of a HPV-specific peptide for the production of a HPV vaccine wherein said peptide is a fragment of a mammalian cell protein that has been degraded by human papillomavirus oncoproteins and which is presented, in combination with HLA, at the surface of the transformed or transfected HPV cell whereby the recognition of this peptide HLA complex by a cytotoxic T lymphocyte results in the elicitation of an immune response.

The invention will now be described by way of the following examples and with reference to the following Figures wherein:

FIG. 1 shows a mechanism for T cell recognition of host cell proteins in cells transformed by human papillomavirus;

FIG. 2 shows a difference in presentation of host cell peptides in normal and HPV transformed cells;

FIG. 3 shows HLA-A2 binding of host protein derived peptides. Specifically, HLA-A*0201 expression of T2 cells was monitored by flow cytometry after overnight incubation with 100 μg test peptides. Each peptide was tested in quadruplicate. A % increase in HLA-A*0201 expression of above 50% was considered significant;

FIG. 4 shows generation of T cell responses in vivo against a peptide derived from human Rb protein. Specifically, HLA-A2/Kb transgenic mice were immunised with 100 μg of test peptide emulsified in incomplete freunds adjuvant. Two to four mice were tested for each peptide. Ten to eleven days later, mice were sacrificed and splenocytes tested in ELISPOT assays. These measured the numbers of IFN⁻ producing T cells (spots) specific for the immunising peptide. Positive results were confirmed in at least two further repeat experiments. Representative data from 1 experiment testing M1₅₈₋₆₆, Rb1, Rb7 and BAK18 peptides, are shown;

FIG. 5 shows CD8⁺ T cells recognising Rb7 can be detected in healthy HLA-A2⁺ donors. Specifically, CD8+ T cells were cultured with Rb7 (panels C&D) or Melan-A/Mart1₂₆₋₃₅ (panels A&B) peptides for 14 days On day 0 and day 14 days, the numbers of peptide specific CD8⁺ T cells were measured using appropriate fluoresceinated peptide:HLA-A2 pentamers. These were analysed on a flow cytometer and expressed as % of gated cells, excluding dead and CD14⁺ cells. Results for donor 5 (from table 4) are shown; and

FIG. 6 shows functional CD8⁺ T cells recognising Rb7 can be detected in healthy donors. Specifically, peripheral blood lymphocytes from HLA-A2⁺ healthy donors were enriched for CD8+ T cells, then cultured for 14 days with Rb7 peptide and antigen presenting cells (APC). The cultured cells were harvested and tested in enzyme linked immunospot (ELISPOT) assays to measure the numbers of T cells able to secrete IFN—in response to Rb7 peptide. Three (A, B, C) out of seven donors were capable of making significant responses (number of spots for T cells+Rb7+PBMC >2 standard deviations above T cells+PBMC).

EXAMPLE 1

Candidate 9 or 10 amino acid peptides from 15 proteins were selected for analysis, see Table 4 for the full list of peptides. One hundred and two peptides predicted to bind to HLA-A*0201 were selected according to published algorithms (33,34). (Table 4). The algorithm we used has been used previously to successfully predict other tumour-specific CTL epitopes (17). We chose HLA-A*0201 as the co-presenting peptide as this is the most common HLA allele (˜40%) among Caucasians, and there are well-defined in vivo and in vitro model systems to facilitate proof of concept experiments. From the initial list of 102 peptides, 62 were synthesized for testing.

Peptides were Tested for Binding to HLA-A*0201.

CTL recognise peptides bound to HLA class I molecules on the cell surface. Therefore peptides must demonstrate binding to HLA to be useful. This can be measured by using a cell based assay measuring an increase in HLA-A*0201 expression resulting from binding.

A cell-based peptide binding assay ((35)) was used to show that the majority (43162) of candidate peptides could bind to HLA-A*0201 (Table 3 FIG. 3).

HLA-A2 Binding of Host Protein Derived Peptides.

HLA-A*0201 expression of T2 cells was monitored by flow cytometry after overnight incubation with 100 μg test peptides. Each peptide was tested in quadruplicate. A percentage increase in HLA-A*0201 expression of above 50% was considered significant.

The level of binding observed could be classified as either strong or moderate, and was comparable to two well-known HLA-A*0201 restricted CTL epitopes (influenza M1 and EBV BMFL1)(FIG. 3).

43 of the peptides that showed a greater than 50% increase in HLA-A*0201 expression were chosen as candidates for testing in immunogenicity experiments.

Testing In Vivo Immunogenicity in Mice.

We have used HLA-A*0201 transgenic mice to test potential human vaccines (36). These mice can be immunised with peptides together with adjuvant to monitor development of in vivo responses. These responses were detected using ELISPOT assays to measure the numbers of IFN-γ securing peptide specific T cells in the spleen.

This was done with 20 peptides from the Rb(6), Muppl (7), BAK (3), DLG (2), AP (2) proteins. All peptides were chosen on the strength of peptide binding (FIG. 3). The M1₅₈₋₆₆ peptide from influenza, a known HLA-A*0201 binder was used as a positive control. Significant T cell responses were seen against the positive control M1₅₈₋₆₆ peptide (7/12 mice) and a peptide from Rb₄₈₅₋₄₉₃ (Rb7) protein (7/10 mice). The results are shown in FIG. 4.

Testing In Vitro Immunogenicity using Human T Lymphocytes.

The candidate peptides must be capable of activating human T lymphocytes that can recognise and kill cancer cells. We detected peptide specific T cells in the peripheral blood of patients with cervical cancer. This proved the concept that such peptides can be immunogenic despite being derived from “self” proteins.

Detection of Peptide Specific T Cells

T cells reactive against peptides of the invention should be preferentially found in HLA-A2⁺ patients with cervical cancer. Blood samples from 4 patients (3 with cervical cancer, 1 with premalignant disease, CIN3) were tested for the presence of CD8⁺ T cells recognising Rb7 peptide. Fluoresceinated multimeric HLA-A2/peptide complexes were used to measure numbers of peptide specific T cells by flow cytometry (10). This assay demonstrated that T cells specific for Rb7 peptide could be detected at low frequency in the blood of 2 patients with cervical cancer. These frequencies were similar to those previously obtained for HPV16 E7 peptide specific T cells (10). This suggests that it should be possible to isolate and propagate Rb specific T cells for further experiments.

Using the technique described above, T lymphocytes recognising certain “self” tumour antigens such as melan-A can also be readily detected in healthy donors (50). However it is usually the case that T lymphocytes recognising “self” antigens are difficult to detect in healthy donors. Blood samples from 8 healthy donors (all HLA-A2+) were tested for the presence of CD8⁺ T cells recognising Rb7 peptide and melan-A peptide, using appropriate fluoresceinated multimers (pentamers). T lymphocytes recognising Rb7 peptide could be detected at variable but generally low frequencies in all 8 healthy donors (Table 6). By contrast T lymphocyte responses against melan-A were extremely high frequency, confirming previous reports (50). The highest frequency of T lymphocytes specific for Rb7 was found in donor 5 (FIG. 5).

The assays described above demonstrate that human T cells recognising Rb7 can be detected in both patients and healthy donors. ELISPOT assays were used to determine whether Rb7 peptide specific CD8⁺ T lymphocytes cultured from healthy donors, were capable of secreting IFN-γ. Seven healthy donors were tested, with 3 donors demonstrating detectable numbers of IFN-γ secreting T lymphocytes (FIG. 5).

Overall these results suggest that host cell protein fragments produced by HPV processing, such as the Rb7 peptide, is immunogenic for human CD8⁺ T lymphocytes, and can elicit functional (IFN-γ secretion) responses. This suggests that it should be possible to isolate and propagate large numbers of Rb specific T cells for either experimental or clinical therapeutic use.

Full List of HLA Class I alleles assigned as of July 2005 HLA-A HLA-B HLA-C HLA-E HLA-F HLA-G A*01010101 B*070201 Cw*010201 E*01010101 F*010101 G*010101 A*01010102N B*070202 Cw*010202 E*01010102 F*010102 G*010102 A*010102 B*070203 Cw*010203 E*01030101 G*010103 A*010103 B*070204 Cw*0103 E*01030102 G*010104 A*0102 B*0703 Cw*0104 E*010302 G*010105 A*0103 B*0704 Cw*0105 E*010303 G*010106 A*0104N B*0705 Cw*0106 E*010304 G*010107 A*0106 B*0706 Cw*0107 E*0104 G*010108 A*0107 B*0707 Cw*0108 G*0102 A*0108 B*0708 Cw*0109 G*0103 A*0109 B*0709 Cw*0110 G*010401 A*0110 B*0710 Cw*0111 G*010402 A*0111N B*0711 Cw*020201 G*010403 A*0112 B*0712 Cw*020202 G*0105N A*0113 B*0713 Cw*020203 G*0106 A*0114 B*0714 Cw*020204 A*0115N B*0715 Cw*020205 A*02010101 B*0716 Cw*0203 A*02010102L B*0717 Cw*0204 A*020102 B*0718 Cw*0205 A*020103 B*0719 Cw*0206 A*020104 B*0720 Cw*0207 A*020105 B*0721 Cw*0208 A*020106 B*0722 Cw*0209 A*020107 B*0723 Cw*0210 A*020108 B*0724 Cw*0211 A*020109 B*0725 Cw*0212 A*020110 B*0726 Cw*030201 A*020111 B*0727 Cw*030202 A*0202 B*0728 Cw*030301 A*020301 B*0729 Cw*030302 A*020302 B*0730 Cw*030303 A*0204 B*0731 Cw*030304 A*0205 B*0732 Cw*030401 A*020601 B*0733 Cw*030402 A*020602 B*0734 Cw*030403 A*020603 B*0735 Cw*0305 A*0207 B*0736 Cw*0306 A*0208 B*0737 Cw*0307 A*0209 B*0738 Cw*0308 A*0210 B*0739 Cw*0309 A*0211 B*0740 Cw*0310 A*0212 B*0741 Cw*0311 A*0213 B*0742 Cw*0312 A*0214 B*0743 Cw*0313 A*0215N B*080101 Cw*0314 A*0216 B*080102 Cw*0315 A*021701 B*0802 Cw*0316 A*021702 B*0803 Cw*0317 A*0218 B*0804 Cw*0318 A*0219 B*0805 Cw*0319 A*022001 B*0806 Cw*04010101 A*022002 B*0807 Cw*04010102 A*0221 B*0808N Cw*040102 A*0222 B*0809 Cw*040103 A*0224 B*0810 Cw*0403 A*0225 B*0811 Cw*040401 A*0226 B*0812 Cw*040402 A*0227 B*0813 Cw*0405 A*0228 B*0814 Cw*0406 A*0229 B*0815 Cw*0407 A*0230 B*0816 Cw*0408 A*0231 B*0817 Cw*0409N A*0232N B*0818 Cw*0410 A*0233 B*0819N Cw*0411 A*0234 B*0820 Cw*0412 A*023501 B*0821 Cw*0413 A*023502 B*0822 Cw*0414 A*0236 B*0823 Cw*0415 A*0237 B*0824 Cw*0416 A*0238 B*0825 Cw*0417 A*0239 B*1301 Cw*050101 A*0240 B*130201 Cw*050102 A*0241 B*130202 Cw*0502 A*0242 B*1303 Cw*0503 A*0243N B*1304 Cw*0504 A*0244 B*1306 Cw*0505 A*0245 B*1307N Cw*0506 A*0246 B*1308 Cw*0507N A*0247 B*1309 Cw*0508 A*0248 B*1310 Cw*0509 A*0249 B*1311 Cw*0510 A*0250 B*1312 Cw*0511 A*0251 B*1313 Cw*0602 A*0252 B*1401 Cw*0603 A*0253N B*140201 Cw*0604 A*0254 B*140202 Cw*0605 A*0255 B*1403 Cw*0606 A*0256 B*1404 Cw*0607 A*0257 B*1405 Cw*0608 A*0258 B*140601 Cw*0609 A*0259 B*140602 Cw*0610 A*0260 B*1407N Cw*0611 A*0261 B*15010101 Cw*0612 A*0262 B*15010102N Cw*0613 A*0263 B*150102 Cw*070101 A*0264 B*150103 Cw*070102 A*0265 B*150104 Cw*070103 A*0266 B*150105 Cw*07020101 A*0267 B*1502 Cw*07020102 A*0268 B*1503 Cw*07020103 A*0269 B*1504 Cw*0703 A*0270 B*1505 Cw*070401 A*0271 B*1506 Cw*070402 A*0272 B*1507 Cw*0705 A*0273 B*1508 Cw*0706 A*027401 B*1509 Cw*0707 A*027402 B*1510 Cw*0708 A*0275 B*151101 Cw*0709 A*0276 B*151102 Cw*0710 A*0277 B*1512 Cw*0711 A*0278 B*1513 Cw*0712 A*0279 B*1514 Cw*0713 A*0280 B*1515 Cw*0714 A*0281 B*1516 Cw*0715 A*0282N B*15170101 Cw*0716 A*0283N B*15170102 Cw*0717 A*0284 B*151702 Cw*0718 A*0285 B*1518 Cw*0719 A*0286 B*1519 Cw*0720 A*03010101 B*1520 Cw*0721 A*03010102N B*1521 Cw*0722 A*03010103 B*1523 Cw*0723 A*030102 B*1524 Cw*0724 A*030103 B*1525 Cw*0725 A*030104 B*1526N Cw*0726 A*0302 B*1527 Cw*0727 A*0303N B*1528 Cw*0728 A*0304 B*1529 Cw*0729 A*0305 B*1530 Cw*0730 A*0306 B*1531 Cw*080101 A*0307 B*1532 Cw*080102 A*0308 B*1533 Cw*0802 A*0309 B*1534 Cw*0803 A*0310 B*1535 Cw*0804 A*0311N B*1536 Cw*0805 A*0312 B*1537 Cw*0806 A*0313 B*1538 Cw*0807 A*0314 B*1539 Cw*0808 A*0315 B*1540 Cw*0809 A*0316 B*1542 Cw*0810 A*0317 B*1543 Cw*0811 A*110101 B*1544 Cw*0812 A*110102 B*1545 Cw*120201 A*110103 B*1546 Cw*120202 A*110104 B*1547 Cw*120203 A*110105 B*1548 Cw*120301 A*1102 B*1549 Cw*120302 A*1103 B*1550 Cw*120303 A*1104 B*1551 Cw*120401 A*1105 B*1552 Cw*120402 A*1106 B*1553 Cw*1205 A*1107 B*1554 Cw*1206 A*1108 B*1555 Cw*1207 A*1109 B*1556 Cw*1208 A*1110 B*1557 Cw*1209 A*1111 B*1558 Cw*1210 A*1112 B*1560 Cw*1211 A*1113 B*1561 Cw*1212 A*1114 B*1562 Cw*1213 A*1115 B*1563 Cw*1214 A*1116 B*1564 Cw*1215 A*1117 B*1565 Cw*140201 A*1118 B*1566 Cw*140202 A*1119 B*1567 Cw*140203 A*1120 B*1568 Cw*140204 A*1121N B*1569 Cw*1403 A*1122 B*1570 Cw*1404 A*1123 B*1571 Cw*1405 A*2301 B*1572 Cw*1406 A*2302 B*1573 Cw*1407N A*2303 B*1574 Cw*150201 A*2304 B*1575 Cw*150202 A*2305 B*1576 Cw*1503 A*2306 B*1577 Cw*1504 A*2307N B*1578 Cw*150501 A*2308N B*1579N Cw*150502 A*2309 B*1580 Cw*150503 A*2310 B*1581 Cw*150504 A*2311N B*1582 Cw*1506 A*2312 B*1583 Cw*1507 A*24020101 B*1584 Cw*1508 A*24020102L B*1585 Cw*1509 A*240202 B*1586 Cw*1510 A*240203 B*1587 Cw*1511 A*240204 B*1588 Cw*1512 A*240205 B*1589 Cw*1513 A*240206 B*1590 Cw*1514 A*240301 B*1591 Cw*160101 A*240302 B*1592 Cw*160102 A*2404 B*1593 Cw*1602 A*2405 B*1594N Cw*160401 A*2406 B*1595 Cw*1606 A*2407 B*1596 Cw*1607 A*2408 B*1597 Cw*1701 A*2409N B*1598 Cw*1702 A*2410 B*1599 Cw*1703 A*2411N B*180101 Cw*1801 A*2413 B*180102 Cw*1802 A*2414 B*1802 A*2415 B*1803 A*2417 B*1804 A*2418 B*1805 A*2419 B*1806 A*2420 B*1807 A*2421 B*1808 A*2422 B*1809 A*2423 B*1810 A*2424 B*1811 A*2425 B*1812 A*2426 B*1813 A*2427 B*1814 A*2428 B*1815 A*2429 B*1817N A*2430 B*1818 A*2431 B*1819 A*2432 B*1820 A*2433 B*2701 A*2434 B*2702 A*2435 B*2703 A*2436N B*270401 A*2437 B*270402 A*2438 B*270502 A*2439 B*270503 A*2440N B*270504 A*2441 B*270505 A*2442 B*270506 A*2443 B*270507 A*2444 B*270508 A*2445N B*270509 A*2446 B*2706 A*2447 B*2707 A*2448N B*2708 A*2449 B*2709 A*2450 B*2710 A*2451 B*2711 A*2452 B*2712 A*2453 B*2713 A*250101 B*2714 A*250102 B*2715 A*2502 B*2716 A*2503 B*2717 A*2504 B*2718 A*260101 B*2719 A*260102 B*2720 A*260103 B*2721 A*260104 B*2723 A*2602 B*2724 A*2603 B*2725 A*2604 B*2726 A*2605 B*2727 A*2606 B*2728 A*260701 B*2729 A*260702 B*2730 A*2608 B*350101 A*2609 B*350102 A*2610 B*350103 A*2611N B*350104 A*2612 B*350201 A*2613 B*350202 A*2614 B*3503 A*2615 B*350401 A*2616 B*350402 A*2617 B*3505 A*2618 B*3506 A*2619 B*3507 A*2620 B*3508 A*2621 B*350901 A*2622 B*350902 A*2623 B*3510 A*2624 B*3511 A*2625N B*3512 A*2626 B*3513 A*29010101 B*351401 A*29010102N B*351402 A*290201 B*3515 A*290202 B*3516 A*290203 B*3517 A*2903 B*3518 A*2904 B*3519 A*2905 B*3520 A*2906 B*3521 A*2907 B*3522 A*2908N B*3523 A*2909 B*3524 A*2910 B*3525 A*2911 B*3526 A*2912 B*3527 A*2913 B*3528 A*2914 B*3529 A*300101 B*3530 A*300102 B*3531 A*300201 B*3532 A*300202 B*3533 A*300203 B*3534 A*3003 B*3535 A*3004 B*3536 A*3006 B*3537 A*3007 B*3538 A*3008 B*3539 A*3009 B*3540N A*3010 B*3541 A*3011 B*3542 A*3012 B*3543 A*3013 B*3544 A*3014L B*3545 A*3015 B*3546 A*310102 B*3547 A*3102 B*3548 A*3103 B*3549 A*3104 B*3550 A*3105 B*3551 A*3106 B*3552 A*3107 B*3553N A*3108 B*3554 A*3109 B*3555 A*3110 B*3556 A*3111 B*3557 A*3112 B*3558 A*3201 B*3559 A*3202 B*3560 A*3203 B*3561 A*3204 B*3701 A*3205 B*3702 A*3206 B*3703N A*3207 B*3704 A*3208 B*3705 A*3209 B*3706 A*3210 B*3707 A*3301 B*3708 A*330301 B*3709 A*330302 B*3801 A*3304 B*380201 A*3305 B*380202 A*3306 B*3803 A*3307 B*3804 A*3308 B*3805 A*3401 B*3806 A*3402 B*3807 A*3403 B*3808 A*3404 B*3809 A*3405 B*3810 A*3406 B*3811 A*3601 B*39010101 A*3602 B*39010102L A*3603 B*390103 A*3604 B*390104 A*4301 B*390201 A*6601 B*390202 A*6602 B*3903 A*6603 B*3904 A*6604 B*3905 A*680101 B*390601 A*680102 B*390602 A*680103 B*3907 A*6802 B*3908 A*680301 B*3909 A*680302 B*3910 A*6804 B*3911 A*6805 B*3912 A*6806 B*391301 A*6807 B*391302 A*6808 B*3914 A*6809 B*3915 A*6810 B*3916 A*6811N B*3917 A*6812 B*3918 A*6813 B*3919 A*6814 B*3920 A*6815 B*3922 A*6816 B*3923 A*6817 B*3924 A*6818N B*3925N A*6819 B*3926 A*6820 B*3927 A*6821 B*3928 A*6822 B*3929 A*6823 B*3930 A*6824 B*3931 A*6825 B*3932 A*6826 B*3933 A*6827 B*3934 A*6828 B*400101 A*6901 B*400102 A*7401 B*400103 A*7402 B*400104 A*7403 B*400105 A*7404 B*400201 A*7405 B*400202 A*7406 B*400203 A*7407 B*4003 A*7408 B*4004 A*7409 B*4005 A*7410 B*40060101 A*7411 B*40060102 A*8001 B*400602 B*4007 B*4008 B*4009 B*4010 B*4011 B*4012 B*4013 B*401401 B*401402 B*401403 B*4015 B*4016 B*4018 B*4019 B*4020 B*4021 B*4022N B*4023 B*4024 B*4025 B*4026 B*4027 B*4028 B*4029 B*4030 B*4031 B*4032 B*4033 B*4034 B*4035 B*4036 B*4037 B*4038 B*4039 B*4040 B*4042 B*4043 B*4044 B*4045 B*4046 B*4047 B*4048 B*4049 B*4050 B*4051 B*4052 B*4053 B*4054 B*4055 B*4056 B*4057 B*4058 B*4059 B*4060 B*4061 B*4101 B*4102 B*4103 B*4104 B*4105 B*4106 B*4107 B*4201 B*4202 B*4204 B*420501 B*420502 B*4206 B*44020101 B*44020102S B*440202 B*440203 B*440301 B*440302 B*4404 B*4405 B*4406 B*4407 B*4408 B*4409 B*4410 B*4411 B*4412 B*4413 B*4414 B*4415 B*4416 B*4417 B*4418 B*4419N B*4420 B*4421 B*4422 B*4423N B*4424 B*4425 B*4426 B*4427 B*4428 B*4429 B*4430 B*4431 B*4432 B*4433 B*4434 B*4435 B*4436 B*4437 B*4438 B*4439 B*4440 B*4441 B*4442 B*4501 B*4502 B*4503 B*4504 B*4505 B*4506 B*4507 B*4601 B*4602 B*4603 B*4604 B*4605 B*47010101 B*47010102 B*4702 B*4703 B*4704 B*4705 B*4801 B*4802 B*4803 B*4804 B*4805 B*4806 B*4807 B*4808 B*4809 B*4810 B*4811 B*4812 B*4813 B*4901 B*4902 B*4903 B*4904 B*5001 B*5002 B*5004 B*510101 B*510102 B*510103 B*510104 B*510105 B*510106 B*510107 B*510201 B*510202 B*5103 B*5104 B*5105 B*5106 B*5107 B*5108 B*5109 B*5110 B*5111N B*5112 B*511301 B*511302 B*5114 B*5115 B*5116 B*5117 B*5118 B*5119 B*5120 B*5121 B*5122 B*5123 B*5124 B*5126 B*5127N B*5128 B*5129 B*5130 B*5131 B*5132 B*5133 B*5134 B*5135 B*5136 B*5137 B*5138 B*520101 B*520102 B*520103 B*520104 B*5202 B*5203 B*5204 B*5205 B*5206 B*5207 B*5208 B*530101 B*530102 B*530103 B*5302 B*5303 B*5304 B*5305 B*5306 B*5307 B*5308 B*5309 B*5310 B*5401 B*5402 B*5403 B*5404 B*5405N B*5406 B*5407 B*550101 B*550102 B*5502 B*5503 B*5504 B*5505 B*5507 B*5508 B*5509 B*5510 B*5511 B*5512 B*5513 B*5514 B*5515 B*5516 B*5517 B*5518 B*5519 B*5601 B*5602 B*5603 B*5604 B*560501 B*560502 B*5606 B*5607 B*5608 B*5609 B*5610 B*5611 B*5612 B*5613 B*5614 B*5615 B*5616 B*570101 B*570102 B*570103 B*5702 B*570301 B*570302 B*5704 B*5705 B*5706 B*5707 B*5708 B*5709 B*5801 B*5802 B*5804 B*5805 B*5806 B*5807 B*5808 B*5809 B*5810N B*5811 B*5901 B*670101 B*670102 B*6702 B*7301 B*7801 B*780201 B*780202 B*7803 B*7804 B*7805 B*8101 B*8102 B*8201 B*8202 B*8301 B*9501 B*9502 B*9503 B*9504

TABLE 2 Monoclonal antibodies used to treat cancer, their target and mechanism of action. Name of monoclonal antibody Target/Mechanism of action Rituximab (Rituxan ®) Used to treat B-cell lymphomas, acts by binding to the CD20 molecule present on all B cells. Herceptin ® Binds to HER2, a growth factor receptor found on some tumour cells, such as breast cancers and lymphomas. Alemtuzumab (MabCampath ®) Used to treat chronic lymphocytic leukaemia. Binds to the CD52 molecule found on all white blood cells. Lym-1 (Oncolym ®) Used to treat lymphoma. Binds to the histocompatibility antigen found on lymphoma cells. Bevacizumab (Avastin ®) Used to treat tumours. Binds to vascular endothelial growth factor, which is found in healthy as well as malignant cells. Cetuximab (Erbitux ®) Used to treat colorectal cancers. Binds to epidermal growth factor receptor, which is also found on normal epithelial cells. Telomerase antibody Used to treat cancers in general. Binds to telomerase, which is the protein responsible for cancer cells over-riding the usual apoptosis mechanisms. This protein is also present on normal cells.

TABLE 3 Host cell proteins known to be targeted for degradation following binding to HPV E6 or E7 proteins. Host cell HPV Gene Accession target* protein Other names name number Function Location Reference Retinoblastoma 1 E7 Retinoblastoma RB1 M15400 cell cycle Nuclear (19) susceptibility regulation, protein tumour suppressor E6AP E6 Human UBE3A AF002224 proteolysis; Cytosolic (24) papillomavirus E6 binds to associated HPV16&18 protein E6 to target (E6AP), Ubiquitin p53 for protein ligase degradation, E3A (UBE3A) can also target itself for ubiquination and degradation C-MYC E6 v-myc MYC AY214166 cell cycle, Nuclear (25) myelocytomatosis gene viral oncogene transcription homolog (avian), myc proto- oncogene product HSCRIB E6 scribbled SCRIB AY062238 cell cycle membrane (26) homologue, vartul HMCM7 E6 minichromosome MCM7 AB004270 DNA nuclear (27) maintenance replication deficient 7, CDC47 MAGI1 E6 membrane MAGI1 AB010894 Signalling, membrane (28) associated tight guanylate kinase, junctions, WW and PDZ cell domain adhesion containing 1, BAIAP1, BAP1, MAGI-1, AIP3 SIPA1L1 E6 signal-induced SIPA1L1 AB007900 GTPase membrane (29) proliferation- activating associated 1 like protein 1, E6 targetted protein 1 (E6TP1) DLG1 E6 discs, large DLG1 U13897 cell growth, membrane (30) homolog 1 adhesion, (DLG1, HDLG), signalling synapse associated protein 97 (SAP97), hdlg BAK E6 BCL2- BAK1 U23765 pro- membrane (31) antagonist/killer 1 apoptotic, in presence of appropriate stimuli binds to BCL-2 to accelerate cell death MPDZ E6 multiple PDZ MPDZ AF093419 tight membrane (32) domain protein junction (MUPP1) formation MGMT E6 O-6- MGMT M29971 DNA repair nuclear (33) methylguanine- DNA methyltransferase MAGI-2 E6 membrane MAGI2 AB014605 same as membrane (34) associated magi-1 guanylate kinase, WW and PDZ domain containing 2, activin receptor interacting protein 1 (ARIP1) MAGI-3 E6 membrane MAGI3 AF213259 same as membrane (34) associated magi-1 guanylate kinase, WW and PDZ domain containing 3 Tuberin E6 tuberous TSC2 AB014460 tumour membrane (35) sclerosis 2 suppressor, (TSC2) GTPase signalling N-MYC E6 v-myc MYCN M13228 tumour membrane (25) myelocytomatosis suppressor viral related oncogene, neuro- blastoma derived (avian), N-MYC proto-oncogene protein *Proteins are listed by their agreed standard names, rather than the published names.

TABLE 4 Host cell proteins and HLA-A*0201 binding peptides Starting SEQ ID Protein Pos Sequence T0.5 NO. Retinoblastoma 563 WLSDsPLFDL 5375 1 Retinoblastoma 475 KLLNdNIFHM 858 2 Retinoblastoma 75 WLTWeKVSSV 736 3 Retinoblastoma 646 SLSLFYKKV 396 4 Retinoblastoma 447 KLGVrLYYRV 365 5 Retinoblastoma 218 LMLCvLDYFI 261 6 Retinoblastoma 485 SLLACALEV 257 7 Retinoblastoma 157 VLFALFSKL 255 8 Retinoblastoma 648 SLFYkKVYRL 182 9 Retinoblastoma 824 KMTPrSRILV 176 10 Retinoblastoma 679 IIWTIFQHTL 157 11 Retinoblastoma 188 ALVLKVSWI 132 12 Retinoblastoma 76 LTWEKVSSV 129 13 Retinoblastoma 703 IMMCSMYGI 109 14 Retinoblastoma 900 KLAEmTSTRT 107 15 Retinoblastoma 219 MLCVLDYFI 98 16 BAK 150 FLGQvTRFVV 761 17 BAK 129 ALLGFGYRL 300 18 BAK 188 ILNVIVVLGV 272 19 BAK 182 NLGNgPILNV 160 20 BAK 195 VVLLGQFVV 91 21 BAK 191 VLVVIGVVLL 84 22 BAK 145 GLTGFLGQV 79 23 DLG 355 ALYDRLADV 2099 24 DLG 275 NLHGvFVAEV 608 25 DLG 345 GLPGDSFYI 333 26 DLG 148 QLLEfNGINL 324 27 DLG 666 VLWIPACPL 301 28 DLG 497 VLILgPLLDV 272 29 DLG 301 LILEyGSLDV 247 30 DLG 480 SLAYqRVQKV 160 31 DLG 498 LILGPLLDV 138 32 DLG 178 ILAQYNPHV 118 33 E6AP 127 YLTEeKVYEI 453 34 E6AP 449 FINEPLNEV 415 35 E6AP 748 YLFRpEEIEL 364 36 E6AP 657 VLYQSLKDL 267 37 E6AP 785 VLIREFWEI 253 38 E6AP 226 KLGPDDVSV 243 39 E6AP 283 YLNLFIIGM 200 40 E6AP 785 VLIReFWEIV 153 41 E6AP 477 FMTCpFILNA 144 42 E6AP 262 YLSPnVECDL 98 43 C-MYC 133 CMWSgFSAAA 113 44 C-MYC 68 GLCSpCYVAV 104 45 C-MYC 177 YLQDLSAAA 94 46 C-MYC 133 CMWSGFSAA 57 47 MUPP1 745 LLPGdRLMFV 1496 48 MUPP1 692 AMWEaGIQHI 590 49 MUPP1 607 LLGENHQDV 485 50 MUPP1 981 YLLEQSSLA 347 51 MUPP1 1650 LLGAiIIHEV 272 52 MUPP1 814 GLADkPLFRA 272 53 MUPP1 606 TLLGeNHQDV 257 54 MUPP1 1547 SLLKtAKMTV 257 55 MUPP1 1772 ILMVNGEDV 214 56 MUPP1 1524 KVGDQILAV 201 57 MUPP1 1766 LMQGdQILMV 196 58 MUPP1 592 KLFSGDELL 136 59 MUPP1 87 TLQNESFLL 124 60 MUPP1 312 GMSSeQVAQV 116 61 MUPP1 751 LMFVNDVNL 97 62 E6TP1 855 SMGAIVWAV 867 63 E6TP1 886 VLIEgETKSV 485 64 E6TP1 960 GLGQLCFHV 403 65 E6TP1 877 LLGIsNEFIV 281 66 E6TP1 1009 QMIDILRTSV 206 67 E6TP1 1592 VLFSsTYPSL 202 68 E6TP1 783 FLLAKVINA 194 69 E6TP1 45 SLGSsVMAPV 160 70 E6TP1 641 FLQLLGERV 157 71 E6TP1 339 ILFDLNEAI 132 72 E6TP1 1522 KLIDLESPT 107 73 MAGI-1 69 LLLEvQCVRV 1794 74 MAGI-1 262 TLQEtALPPV 656 75 MAGI-1 1054 KVGDRILAV 201 76 MAGI-1 161 FLTVkEFLDL 187 77 MAGI-1 80 GLPRyDVLGV 160 78 MAGI-1 717 LLVQrGCLPV 118 79 MAGI-1 527 VLGHTHAQV 118 80 MCM7 356 RLAQHITYV 880 81 MCM7 335 LLSRfDLLWL 459 82 MCM7 60 KMQEHSDQV 353 83 MCM7 259 VLADgGVCCI 167 84 MCM7 439 ALARIRMVDV 160 85 MCM7 381 KLMRrYIAMC 148 86 MCM7 178 LLLLLVGGV 131 87 MCM7 481 ALDEyEELNV 114 88 MCM7 273 KMAEaDRTAI 108 89 Vartul 713 KLLEvNGVAL 1134 90 Vartul 99 SLPAsLSFLV 403 91 Vartul 348 YLLPQQPPL 364 92 Vartul 229 ILTEnLLMAL 342 93 Vartul 244 KLTKITNLNV 243 94 Vartul 15 FMQLVELDV 231 95 Vartul 1199 KLDYrALAAV 224 96 Vartul 68 ALNDvSLQAL 201 97 Vartul 63 SLAHIALNDV 160 98 Vartul 992 ILAVNGQDV 118 99 Vartul 849 VLSINGVDV 118 100 Vartul 129 ALPNIRELWL 117 101 MGMT 98 VLWKLLKVV 925 102 MGMT 161 GLAVKEWLL 160 103 MGMT 167 WLLAhEGHRL 364 104 MGMT 89 FQQEsFTRQV 101 105 N-Myc 421 VILKkATEYV 162 106 440 LLLEkEKLQA 128 107 446 KLQArQQQLL 75 108 MAGI-2 65 LLLEvNETPV 1794 109 993 KLIKdAGLSV 243 110 970 KVGDRILAV 201 111 43 YLGEvKPGKV 171 112 76 GLTIrDVLAV 160 113 528 AIMERPPPV 145 114 751 AIYESRQQV 125 115 481 VLGHTHADV 118 116 639 GLCEGDLIV 117 117 65 LLLEvNETPV 1794 118 993 KLIKdAGLSV 243 119 970 KVGDRILAV 201 120 43 YLGEvKPGKV 171 121 76 GLTIrDVLAV 160 122 528 AIMERPPPV 145 123 751 AIYESRQQV 125 124 481 VLGHTHADV 118 125 639 GLCEGDLIV 117 126 MAGI-3 70 VLLEvNGTPV 1794 127 502 FQLVpVNQYV 660 128 503 QLVPVNQYV 383 129 11 WLSKvQECAV 320 130 923 KVGDHISAV 201 131 726 KLDPSEVYL 164 132 654 NLTHIQVVEV 160 133 200 FQPDPVDQV 150 134 1067 NMGLFILRL 135 135 1121 LLLLrPGTGL 134 136 1122 LLLRPGTGL 134 137 499 VQMFQLVPV 102 138 70 VLLEvNGTPV 98 139 502 FQLVpVNQYV 91 140 503 QLVPVNQYV 85 141 11 WLSKvQECAV 79 142 Tuberin 464 KVLDVLSFV 4088 143 176 FLLVLVNLV 2723 144 1060 WLVGnKLVTV 736 145 291 LLRGAVFFV 659 146 1702 IVSDrNLPFV 537 147 80 ALWKAVADL 408 148 1208 WLMSLENPL 364 149 688 LLFRvLLQCL 309 150 117 GVLRaLFFKV 248 151 1065 KLVTVTTSV 243 152 464 KVLDvLSFVL 236 153 155 YLEEeLADFV 226 154 258 KLMRNLLGT 222 155 1033 YVFSNFTAV 197 156 235 SLPLFIVTL 187 157 612 LQAFdFLFLL 187 158 617 FLFLIRADSL 178 159 506 KLATgLLVDL 172 160 360 ILLNIIERL 151 161

TABLE 5 Summary of peptide binding No. peptides Source protein binding BAK 6/7 DLG  7/10 CMYC 2/4 E6AP  9/10 MUPP1 13/15 Retinoblastoma  6/16 Total 43/62

TABLE 6 Frequency of CD8⁺ T cells recognising Rb peptide in healthy volunteers. Peripheral blood lymphocytes from HLA-A2⁺ healthy donors were enriched for CD8⁺ T cells, then cultured for 14 days with Rb7 peptide and antigen presenting cells (APC) or Melan-A (Mart1₂₆₋₃₅) peptide. Cells were harvested and tested with HLA-A2/Rb7 pentamer or HLA-A2/Melan-A pentamer. The numbers of CD8⁺ pentamer⁺ T lymphocytes were measured by flow cytometry, excluding dead and CD14⁺ cells. Frequency of CD8⁺ pentamer⁺ cells Donor Rb Melan A 1 1/2500  1/24 2 1/1000 1/4 3 1/1000 1/3 4 1/909  1/3 5 1/120  1/4 6 1/3333  1/208 7 1/2000 1/3 8 1/2500 1/3

REFERENCES

-   1. Mandelboim, O., E. Vadai, M. Fridkin, A. Katz-Hillel, M.     Feldman, G. Berke, and L. Eisenbach. 1995. Regression of established     murine carcinoma metastases following vaccination with     tumour-associated antigen peptides. Nature Medicine. 1:1179. -   2. Heslop, H. E., and C. M. Rooney. 1997. Adoptive cellular     immunotherapy for EBV lymphoproliferative disease. Immunological     Reviews 157:217. -   3. Overwijk, W. W., D. S. Lee, D. R. Surman, K. R. Irvine, C. E.     Touloukian, C. C. Chan, M. W. Carroll, B. Moss, S. A. Rosenberg,     and N. P. Restifo. 1999. Vaccination with a recombinant vaccinia     virus encoding a ‘self’ antigen induces autoimmune vitiligo and     tumor cell destruction in mice: requirement for CD4(+) T     lymphocytes. Proceedings of the National Academy of Sciences of the     United States of America 96:2982. -   4. Gao, L., I. Bellantuono, A. Elsasser, S. B. Marley, M. Y.     Gordon, J. M. Goldman, and H. J. Stauss. 2000. Selective elimination     of leukemic CD34(+) progenitor cells by cytotoxic T lymphocytes     specific for WT1. Blood 95:2198. -   5. Theobald, M., J. Biggs, D. Dittmer, A. J. Levine, and L. A.     Sherman. 1996. Targeting p53 as a general tumour antigen.     Proceedings of the National Academy of Sciences of the United States     of America 92:11993. -   6. Walboomers, J. M., M. V. Jacobs, M. M. Manos, F. X. Bosch, J. A.     Kummer, K. V. Shah, P. J. Snijders, J. Peto, C. J. Meijer, and N.     Munoz. 1999. Human papillomavirus is a necessary cause of invasive     cervical cancer worldwide. Journal of Pathology 189:12. -   7. Borysiewicz, L. K., A. Fiander, M. Nimako, S. Man, G. W. G.     Wilkinson, D. Westmoreland, A. S. Evans, M. Adams, S. N.     Stacey, M. E. G. Boursnell, E. Rutherford, J. K. Hickling, and S. C.     Inglis. 1996. A recombinant vaccinia virus encoding human     papillomavirus type 16 and type 18, e6 and e7 proteins as     immunotherapy for cervical cancer. Lancet 347:1523. -   8. Nimako, M., A. N. Fiander, G. W. Wilkinson, L. K. Borysiewicz,     and S. Man. 1997. Human papillomavirus-specific cytotoxic T     lymphocytes in patients with cervical intraepithelial neoplasia     grade III. Cancer Res 57:4855. -   9. Evans. E. M., S. Man, A. S. Evans, and L. K. Borysiewicz. 1997.     Infiltration of cervical cancer tissue with human     papillomavirus-specific cytotoxic T-lymphocytes. Cancer Res 57:2943. -   10. Youde, S. J., P. R. Dunbar, E. M. Evans, A. N. Fiander, L. K.     Borysiewicz, V. Cerundolo, and S. Man. 2000. Use of fluorogenic     histocompatibility leukocyte antigen-A*0201/HPV 16 E7 peptide     complexes to isolate rare human cytotoxic T-lymphocyte-recognizing     endogenous human papillomavirus antigens. Cancer Res 60:365. -   11. Frazer, I. H., R. Thomas, J. Zhou, G. R. Leggatt, L. Dunn, N.     McMillan, R. W. Tindle, L. Filgueira, P. Manders, P. Barnard, and M.     Sharkey. 1999. Potential strategies utilised by papillomavirus to     evade host immunity. Immunological Reviews 168:131. -   12. Khammanivong, V., X. S. Liu, W. J. Liu, S. J. Rodda, G. R.     Leggatt, R. W. Tindle, I. H. Frazer, and G. J. Fernando. 2003.     Paucity of functional CTL epitopes in the E7 oncoprotein of cervical     cancer associated human papillomavirus type 16. Immunol Cell Biol     81:1. -   13. Scheffner, M., Munger K, Huibregtse J M, and H. PM. 1992.     Targeted degradation of the retinoblastoma protein by human     papillomavirus E7-E6 fusion proteins. European Molecular Biology     Organisation Journal 11:2425. -   14. Scheffner, M., B. A. Werness, J. M. Huigbretse, A. J. Levine,     and P. M. Howley. 1990. The E6 oncoprotein encoded by human     papillomavirus types 16 and 18 promotes the degradation of p53. Cell     63:1129. -   15. Higashitsuji, H. K. Itoh, T. Nagao, S. Dawson., K. Nonoguchi, T.     Kido, R. J. Mayer, S. Arii, and J. Fujita. 2000. Reduced stability     of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat     protein overexpressed in hepatomas. Nature Medicine 6:96. -   16. Rongcun, Y., F. Salazar-Onfray, J. Charo, K. Malmberg, K.     Evrin, H. Maes, K. Kono, C. Hising, M. Petersson, O. Larsson, L.     Lan, E. Appella, A. Sette, E. Celis, and R. Kiessling. 1999.     Identification of New HER2/neu-Derived Peptide Epitopes That Can     Elicit Specific CTL Against Autologous and Allogeneic Carcinomas and     Melanomas 1. The Journal of Immunology: 163:1037-1044. -   17. Vonderheide, R. H., W. C. Hahn, J. L. Schultze, and L. M.     Nadler. 1999. The telomerase catalytic subunit is a widely expressed     tumor-associated antigen recognized by cytotoxic T lymphocytes.     Immunity 10:673. -   18. Berezutskaya, E., and S. Bagchi. 1997. The human papillomavirus     E7 oncoprotein functionally interacts with the S4 subunit of the 26     S proteasome J Biol Chem 272:30135. -   19. Boyer, S. N., D. E. Wazer, and V. Band. 1996. E7 protein of     human papilloma virus 16 induces degradation of retinoblastoma     protein through the ubiquitin proteasome pathway. Cancer research     56:4620. -   20. Michalek, M. T., E. P. Grant, C. Gramm, A. L. Goldberg,     and K. L. Rock. 1993. A role for the ubiquitin-dependent proteolytic     pathway in MHC class I-restricted antigen presentation. Nature     363:552. -   21. Cerundolo, V., A. Benham, V. Braud, S. Mukherjee, K. Gould, B.     Macino, J. Neefjes, and A. Townsend. 1997. The proteasome-specific     inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte     epitopes in human and murine cells. Eur J Immunol 27:336. -   22. Vierboom, M., S. Zwaveling, G. Bos, M. Ooms, G. Krietemeijer, C.     Melief, and R. Offring a. 2000. High Steady-State Levels of p53 Are     Not a Prerequisite for Tumor Eradication by Wild-Type p53-specific     Cytotoxic T Lymphocytes. Cancer Research 60:5508-5513. -   23. Vierboom, M. P., H. W. Nijman, R. Offring a, E. I. van der     Voort, T. van Hall, L. van den Broek, G. J. Fleuren, P.     Kenemans, W. M. Kast, and C. J. Melief. 1997. Tumor eradication by     wild-type p53-specific cytotoxic T lymphocytes. Journal of     Experimental Medicine 186:695. -   24. Kao, W. H., S. L. Beaudenon, A. L. Talis, J. M. Huibregtse,     and P. M. Howley. 2000. Human Papillomavirus Type 16 E6 Induces     Self-Ubiquitination of the E6AP Ubiquitin-Protein Ligase. J. Virol.     74:6408. -   25. Gross-Mesilaty, S., E. Reinstein, B. Bercovich, K. E. Tobias, A.     L. -   Schwartz, C. Kahana, and A. Ciechanover. 1998. Basal and human     papillomavirus E6 oncoprotein-induced degradation of Myc proteins by     the ubiquitin pathway. Proceedings of the National Academy of     Sciences of the United States of America 95:8058. -   26. Nakagawa, S., and J. M. Huibregtse. 2000. Human scribble     (Vartul) is targeted for ubiquitin-mediated degradation by the     high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein     ligase. Mol Cell Biol 20:8244. -   27. Kuhne, C., and L. Banks. 1998. E3-ubiquitin ligase/E6-AP links     multicopy maintenance protein 7 to the ubiquitination pathway by a     novel motif, the L2G box. J Biol Chem 273:34302. -   28. Glaunsinger, B. A., S. S. Lee, M. Thomas, L. Banks, and R.     Javier. 2000. Interactions of the PDZ-protein MAGI-1 with adenovirus     E4-ORF1 and high-risk papillomavirus E6 oncoproteins. Onicogene     19:5270. -   29. Gao, Q., L. Singh, A. Kumar, S. Srinivasan, D. E. Wazer, and V.     Band. 2001. Human papillomavirus type 16 E6-induced degradation of     E6TP1 correlates with its ability to immortalize human mammary     epithelial cells. J Virol 75:4459. -   30. Pim, D., M. Thomas, R. Javier, D. Gardiol, and L. Banks. 2000.     HPV E6 targeted degradation of the discs large protein: evidence for     the involvement of a novel ubiquitin ligase Oncogene 19:719. -   31. Thomas, M., and L. Banks. 1998. Inhibition of Bak-induced     apoptosis by HPV-18 E6. Oncogene 17:2943. -   32. Lee, S., B. Glaunsinger, F. Mantovani, L. Banks, and R.     Javier. 2000. Multi-PDZ Domain Protein MUPP1 Is a Cellular Target     for both Adenovirus E4-ORF1 and High-Risk Papillomavirus Type 18 E6     Oncoproteins. Journal of Virology 74:9680-9693. -   33. Srivenugopal, K. S., and F. Ali-Osman. 2002. The DNA repair     protein, O(6)-Methylguanine-DNA methyltransferase is a proteolytic     target for the E6 human papillomavirus oncoprotein. Oncogene     21:5940. -   34. Thomas, M., R. Laura, K. Hepner, E. Guccione, C. Sawyers, L.     Lasky, and L. Banks. 2002. Oncogenic human papillomavirus E6     proteins target the MAGI-2 and MAGI-3 proteins for degradation.     Oncogene 21:5088. -   35. Lu, Z., X. Hu, Y. Li, L. Zheng, Y. Zhou, H. Jiang, T. Ning, Z.     Basang, C. Zhang, and Y. Ke. 2004. Human papillomavirus 16 E6     oncoprotein interferences with insulin signaling pathway by binding     to tuberin. J Biol Chem In press. -   36. Parker, K. C., M. A. Bednarek, L. K. Hull, U. Utz, B.     Cunningham, H. J. Zweerink, W. E. Biddison, and J. E. Coligan. 1992.     Sequence motifs important for peptide binding to the human MHC class     I molecule, HLA-A2. Journal of Immunology 149:3580. -   37. Engelhard, V. H. 1994. Structure of peptides associated with MHC     class I molecules. Current Opinion in Immunology 6:13. -   38. Ellis, J. R., P. J. Keating, J. Baird, E. F. Hounsell, D. V.     Renouf, M. Rowe, D. Hopkins, M. F. Duggan-Keen, J. S.     Bartholomew, L. S. Young, and P. L. Stern. 1995. The association of     an HPV16 oncogene variant with HLA-B7 has implications for vaccine     design in cervical cancer. Nature Medicine 1:464. -   39. Faulkner, L., L. K. Borysiewicz, and S. Man. 1998. The use of     human leucocyte antigen class I transgenic mice to investigate human     immune function. J Immunol Methods 221:1. -   40. Dunbar, P., J. Chen, D. Chao, N. Rust, H. Teisserenc, G. Ogg, P.     Romero, P. Weynants, and V. Cerundolo. 1999. Rapid cloning of tumour     specific CTL suitable for adoptive immunotherapy of melanoma.     Journal of Immunology 162:6959. -   41. Rooney, C. Mh, C. A. Smith, C. Y. C. Ng, S. K. Loftin, J. W.     Sixbey, Y. Gan, D. K. Srivastava, L. C. Bowman, R. A. Krance, M. K.     Brenner, and H. E. Heslop. 1998. Infusion of cytotoxic T cells for     the prevention and treatment of epstein-barr virus-induced lymphoma     in allogeneic transplant recipients Blood 92:1549. -   42. Stanislawski, T., R. H. Voss, C. Lotz, E. Sadovnikova, R. A.     Willemsen, J. Kuball, T. Ruppert, R. L. Bolhuis, C. J. Melief, C.     Huber, H. J. Stauss, and M. Theobaid. 2001. Circumventing tolerance     to a human MDM2-derived tumor antigen by TCR gene transfer. Nat     Immunol 2:962. -   43. Shah, K. V. 1998. Do human papillomavirus infections cause oral     cancer? J Natl Cancer Inst 90:1585. -   44. Shamanin, V., H. zur Hausen, D. Layergne, C. M. Proby, I. M.     Leigh, C. -   Neumann, H. Hamm, M. Goos, U.-F. Haustein, E. G. Jung, G. Plewig, H.     Wolff, and E.-M. de Villiers. 1996. Human papillomavirus infections     in nonmelanoma skin cancers from renal transplant recipients and     nonimmunosuppressed patients. Journal of the National Cancer     Institute 88:802. -   45. Kiviat, N. B. 1999, Papillomaviruses in non-melanoma skin     cancer: epidemiological aspects. Semin Cancer Biol 9.:397. -   46. Man, S. and A. Fiander. 2001. Immunology of human papillomavirus     infection in lower genital tract neoplasia. Best Pract Res Clon     Obstet Gynaecol 15:701. -   47. Palefsky, J. M. 1997. Cutaneous and genital HPV-associated     lesions in HIV-infected patients. Clin Dermatol 15:439. -   48. De Luca, A., A. Baldi, V. Esposito, C. M. Howard, L. Bagella, P.     Rizzo, M. Caputi, H. I. Pass, G. G. Giordano, F. Baldi, M. Carbone,     and A. Giordano. 1997. The retinoblastoma gene family pRb/p105,     p107, pRb2/p130 and simian virus-40 large T-antigen in human     mesotheliomas Nature Medicine 3:913. -   49. Li, B., and Q. P. Dou. 2000. Bax degradation by the     ubiquitin/proteasome-dependent pathway: involvement in tumor     survival and progression. Proc Natt Acad Sci USA 97:3850. -   50. Pittet, M. J., Valmori, D., Dunbar, P. R., Speiser, D. E.,     Linard, D., Lejeune, F., Fleischhauer, K., Cerundolo, V.,     Cerottini, J. C., and Romero, P. 1999. High Frequencies of Naive     Melan-A/MART-1-specific CD8(+) T Cells in a Large Proportion of     Human. Histocompatibility Leukocyte Antigen (HLA)-A2 Individuals.     Journal of Experimental Medicine 190:705-716. 

1. A vaccine for treating HPV-associated diseases, comprising: at least one isolated, purified, synthesised or recombinant peptide, wherein the peptide is a fragment of a host cell protein that has been degraded by a human papillomavirus (HPV) oncoprotein, and can elicit a cytotoxic T-lymphocyte (CTL) response when administered to a mammal.
 2. A vaccine according to claim 1, wherein the mammal is human.
 3. A vaccine according to claim 1, wherein the oncoprotein is E6 or E7.
 4. A vaccine according to claim 1, wherein the host cell protein comprises one or more of the proteins shown in Table
 3. 5. A vaccine according to claim 1, wherein the peptide is HPV-specific or tumour-specific, meaning that it is presented in high amounts on the cell surface of HPV transformed or tumour cells, relative to normal cells.
 6. A vaccine according to claim 5, wherein the peptide is 9-30 amino acids in length.
 7. A vaccine according to claim 6, wherein the peptide is 9-11 amino acids in length.
 8. A vaccine according to claim 7, wherein said vaccine comprises one or more of the peptides selected from the peptides shown in Table 4 (SEQ ID NO. 1-161).
 9. A vaccine according to claim 1, wherein the CTL response is HPV-specific or tumour-specific, meaning that the CTL can recognise HPV-transformed cells or tumour cells expressing the peptide of said vaccine.
 10. A vaccine according to claims 1, 3, 4, 5, 8, or 9, which further comprises a major histocompatibility complex molecule.
 11. A vaccine according to claim 10, wherein the major histocompatibility complex molecule is a class I molecule.
 12. A vaccine according to claim 10, wherein the major histo-compatibility complex molecule is a human leucocyte antigen (HLA).
 13. A vaccine according to claim 10, wherein the major histocompatibility complex molecule comprises one or more of those shown in Table
 1. 14. A vaccine for treating HPV-associated diseases, comprising. at least one isolated, purified, synthesised or recombinant nucleic acid molecule encoding at least one peptide selected from the peptides shown in Table 4 (SEQ ID NO. 1-161), and optionally a major histocompatibility complex molecule comprising at least one of the major histocompatibility complex molecules shown in Table
 1. 15. A vaccine according to claim 14, wherein the nucleic acid molecule is in a vector and comprises a recombinant construct.
 16. A vaccine according to claim 15, wherein the construct is adapted for the expression of said peptide or peptide/HLA complex in a selected host system.
 17. A vaccine according to claim 14, wherein the disease is any one of a cervical cancer, a head and neck squamous cell cancer, a non-melanoma skin cancer, a liver cancer, a mesothioloma, or a prostate cancer.
 18. A vector comprising a nucleic acid molecule encoding a peptide selected from the peptides shown in Table 4 (SEQ ID NO. 1-161), and optionally, at least one major histocompatibility complex molecule selected from those listed in Table
 1. 19. A host cell transformed or transfected by a vector according to claim
 18. 20. A vector according to claim 18, wherein said vector is a cell, plasmid, virus, live organism or other vehicle having incorporated expressibly therein a nucleic acid molecule encoding a peptide selected from the peptides shown in Table 4 (SEQ ID NO. 1-161) and optionally, a major histocompatibility complex molecule selected from those listed in Table
 1. 21. A method of manufacturing a vaccine, which method comprises: culturing a host cell transformed or transfected with a vector according to claim 18, and isolating/purifying the resulting construct product.
 22. A method of identifying HPV-specific or tumour-specific CTL, or the CTL receptors thereof, comprising: (a) culturing a sample containing CTL with at least one peptide that represents a fragment of a host cell protein that is degraded by HPV proteins when said host cell is transformed or transfected by HPV, whereby said peptide is ultimately presented on a surface of a virally-infected cell; and (b) selecting CTL that recognise said peptide by binding thereto and, where the CTL receptor is to be identified, determining the nature of the receptor that binds said peptide.
 23. A method according to claim 22, wherein the peptide is selected from the peptides shown in Table 4 (SEQ ID NO. 1-161).
 24. A method according to claim 22 or claim 23, wherein the CTL are CD8⁺ cells.
 25. A peptide, or nucleic acid molecule encoding same, selected from the peptides shown in Table 4 (SEQ ID NO 1-161).
 26. A method of treatment, comprising administering a vaccine according to claim 1 to a mammal to be treated.
 27. The method according to claim 26, wherein the mammal is human. 